This proposal is a renewal of 5R01EY013012-12 Patterning the retina for color vision. Color vision requires the comparison between photoreceptors with different sensitivity. In Drosophila, this is achieved by photoreceptors R7 and R8. 30% of R7s contain UV-sensitive Rh3 and are associated with R8 containing blue-Rh5. The remaining 70% of R7s contain UV-Rh4 associated with R8 containing green-Rh6. This stochastic choice is made in R7 where the transcription factor Spineless activates Rh4. In the absence of spineless, R7s express Rh3 and signal R8 to express Rh5. This signal is then interpreted by a bistable loop between the Hippo/Warts tumor suppressor pathway and the growth regulator Melted. After the R8 fate is established, the Rhodopsin molecules themselves contribute to its maintenance in order to avoid co-expression of other Rhodopsins. In aim 1, we will determine how the architecture of the Hippo/Warts pathway differs in tumor suppression where it is homeostatically regulated to control proliferation, and in R8 fate specification where t is bistable. We will determine when and how the pathway is activated and how positive feedback loops control the R8 decision. We will then analyze the cross-transcriptional repression of warts and melted. In aim 2, we will elucidate the pathway downstream of Rhodopsins that is required for the exclusion mechanisms ensuring that a single Rhodopsin is expressed in a photoreceptor. We will also address how the Rhodopsin activity pathway interacts with the Warts/Melted bistable loop. In aim 3, we will characterize novel regulators of the R8 subtype specification pathway that were identified in an RNAi screen. We will also take advantage of wild type natural variants that exhibit extensive co-expression of Rh5 and Rh6 to identify new factors involved in the exclusion mechanism. Finally, we will extend the RNAi screen to signaling molecules in order to target the pathway involved in the communication between R7 and R8. The results of this study will provide fundamental insights into the role of the Hippo/Warts tumor suppressor pathway in cell specification and will provide insights into sensory receptor cross- regulation. Overall, our findings will not only provide novel fundamental concepts for the field of sensory organ development, but will also contribute to cell fate specification and growth regulation. PUBLIC HEALTH RELEVANCE: This project addresses fundamental questions about retinal patterning and the utilization of a tumor suppressor pathway for tissue specification. It addresses the different architectures of the regulatory network in growth control and in retinal patterning. The proposal will also provide a description of the pathway by which a sensory receptor expressed in a receptor cell is involved in the exclusion of the other receptors in this cell.